Water soluble fluorescent or colored dyes and methods for their use

ABSTRACT

Compounds useful as fluorescent or colored dyes are disclosed. The compounds have the following structure (I): including stereoisomers, salts and tautomers thereof, wherein R 1 , R 2 , R 3 , L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , M 1 , M 2 , A, q, w and n are as defined herein. Methods associated with preparation and use of such compounds are also provided.

BACKGROUND OF THE INVENTION

1. Field

The present invention is directed to novel fluorescent or colored dyesand methods for their preparation and use in various analytical methods.

2. Description of the Related Art

Fluorescent and/or colored dyes are known to be particularly suitablefor applications in which a highly sensitive detection reagent isdesirable. Dyes that are able to preferentially label a specificingredient or component in a sample enable the researcher to determinethe presence, quantity and/or location of that specific ingredient orcomponent. In addition, specific systems can be monitored with respectto their spatial and temporal distribution in diverse environments.

Fluorescence and colorimetric methods are extremely widespread inchemistry and biology. These methods give useful information on thepresence, structure, distance, orientation, complexation and/or locationfor biomolecules. In addition, time-resolved methods are increasinglyused in measurements of dynamics and kinetics. As a result, manystrategies for fluorescence or color labeling of biomolecules, such asnucleic acids and protein, have been developed.

Perylenes and related dyes have high photochemical persistency(chemical, thermal, and photochemical stability) and high fluorescencequanta yield and are used in a variety of reprographic processes, solarcells, photovoltaic devices, and dye lasers. However, perylenederivatives have been used primarily as pigments and fluorescent dyes.Perylene dyes of various colors and light-absorbing properties have beenreported. For example, Becker S. et al, Chem. Eur. J., 6,213,984,(2000), report the synthesis of thermotropic perylenedicarboximidechromophores that show a color change from blue to orange. Perylene andrelated chromophores have seen limited use as biomolecular probes,apparently due to the strongly hydrophobic character of these types ofmolecules and difficulties with regiospecific labeling of biomoleculeswith the same.

There is thus a need in the art for water soluble dyes and biomarkersthat permit visual or fluorescent detection of biomolecules withoutprior illumination or chemical or enzymatic activation. Ideally, suchdyes and biomarkers should be intensely colored or fluorescent andshould be available in a variety of colors and fluorescent wavelengths.The present invention fulfills this need and provides further relatedadvantages.

BRIEF SUMMARY

In brief, the present invention is generally directed to compoundsuseful as water soluble, fluorescent or colored dyes and probes thatenable visual detection of biomolecules and other analytes, as well asreagents for their preparation. Methods for visually detecting abiomolecule and for determining the size of a biomolecule are alsodescribed. The water soluble, fluorescent or colored dyes of theinvention are intensely colored and/or fluorescent and can be readilyobserved by visual inspection or other means. In some embodiments thecompounds may be observed without prior illumination or chemical orenzymatic activation. By appropriate selection of the dye, as describedherein, visually detectable biomolecules of a variety of colors may beobtained.

In one embodiment, compounds having the following structure (I) areprovided:

or a stereoisomer, tautomer or salt thereof, wherein R¹, R², R³, L¹, L²,L³, L⁴, L⁵, L⁶, M¹, M², A, q, w and n are as defined herein.

In another embodiment, a method for staining a sample is provided, themethod comprises adding to said sample a representative compound asdescribed herein in an amount sufficient to produce an optical responsewhen said sample is illuminated at an appropriate wavelength.

In still other embodiments, the present disclosure provides a method forvisually detecting a biomolecule, comprising:

(a) providing a representative compound described herein; and

(b) detecting the compound by its visible properties.

Other disclosed methods include a method for visually detecting abiomolecule, the method comprising:

(a) admixing any of the disclosed compounds with one or morebiomolecules; and

(b) detecting the compound by its visible properties.

Other embodiments are directed to a composition comprising any one ofthe disclosed compounds and one or more biomolecules. Use of suchcomposition in analytical methods for detection of the one or morebiomolecules is also provided.

These and other aspects of the invention will be apparent upon referenceto the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments of theinvention. However, one skilled in the art will understand that theinvention may be practiced without these details.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is, as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

“Amino” refers to the —NH₂ group.

“Carboxy” refers to the —CO₂H group.

“Cyano” refers to the —CN group.

“Formyl” refers to the —C(═O)H group.

“Hydroxy” or “hydroxyl” refers to the —OH group.

“Imino” refers to the ═NH group.

“Nitro” refers to the —NO₂ group.

“Oxo” refers to the ═O substituent group.

“Sulfhydryl” refers to the —SH group.

“Thioxo” refers to the ═S group.

“Alkyl” refers to a straight or branched hydrocarbon chain groupconsisting solely of carbon and hydrogen atoms, which is saturated orunsaturated (i.e., contains one or more double and/or triple bonds),having from one to twelve carbon atoms (C₁-C₁₂ alkyl), preferably one toeight carbon atoms (C₁-C₈ alkyl) or one to six carbon atoms (C₁-C₆alkyl), and which is attached to the rest of the molecule by a singlebond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl),n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl,2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl,penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and thelike. Unless stated otherwise specifically in the specification, analkyl group may be optionally substituted.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a substituentgroup, consisting solely of carbon and hydrogen, which is saturated orunsaturated (i.e., contains one or more double and/or triple bonds), andhaving from one to twelve carbon atoms, e.g., methylene, ethylene,propylene, n-butylene, ethenylene, propenylene, n-butenylene,propynylene, n-butynylene, and the like. The alkylene chain is attachedto the rest of the molecule through a single or double bond and to thesubstituent group through a single or double bond. The points ofattachment of the alkylene chain to the rest of the molecule and to thesubstituent group can be through one carbon or any two carbons withinthe chain. Unless stated otherwise specifically in the specification, analkylene chain may be optionally substituted.

“Alkoxy” refers to a group of the formula —OR_(a) where R_(a) is analkyl group as defined above containing one to twelve carbon atoms.Unless stated otherwise specifically in the specification, an alkoxygroup may be optionally substituted.

“Alkylamino” refers to a group of the formula —NHR_(a) or —NR_(a)R_(a)where each R_(a) is, independently, an alkyl group as defined abovecontaining one to twelve carbon atoms. Unless stated otherwisespecifically in the specification, an alkylamino group may be optionallysubstituted.

“Alkylether” refers to any alkyl group as defined above, wherein atleast one carbon-carbon bond is replaced with a carbon-oxygen bond. Thecarbon-oxygen bond may be on the terminal end (as in an alkoxy group) orthe carbon oxygen bond may be internal (i.e., C—O—C). Alkylethersinclude at least one carbon oxygen bond, but may include more than one.For example, polyethylene glycol (PEG) is included within the meaning ofalkylether. Unless stated otherwise specifically in the specification,an alkylether group may be optionally substituted. For example, in someembodiments and alkylether is substituted with an alcohol or phosphate.

“Alkylenether” refers to any alkylene group as defined above, wherein atleast one carbon-carbon bond is replaced with a carbon-oxygen bond. Thecarbon-oxygen bond may be on the terminal end (as in an alkoxy group) orthe carbon oxygen bond may be internal (i.e., C—O—C). Alkylenethersinclude at least one carbon oxygen bond, but may include more than one.For example, polyethylene glycol (PEG) is included within the meaning ofalkylenether. Unless stated otherwise specifically in the specification,an alkylenether group may be optionally substituted.

“Alkylphospho” refers to the —RP(═O)(R_(a))R_(b) group, wherein R is analkylene group, R_(a) is OH, O⁻ or OR_(c); and R_(b) is —Oalkyl or—Oalkylether, wherein R_(c) is a counter ion (e.g., Na+ and the like).Unless stated otherwise specifically in the specification, analkylphospho group may be optionally substituted. For example, incertain embodiments, the —Oalkyl or —Oalkylether moiety (R_(b)) in aalkylphospho group is optionally substituted with one or more ofhydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether.“Oalkylphospho is an alkylphospho group connected to the remainder ofthe molecule via an oxygen atom. Unless stated otherwise specifically inthe specification, an Oalkylphospho group may be optionally substituted.

“Alkyetherphospho” refers to the —RP(═O)(R_(a))R_(b) group, wherein R isan alkylenether group, R_(a) is OH, O⁻ or OR_(c); and R_(b) is —Oalkylor —Oalkylether, wherein R_(c) is a counter ion (e.g., Na+ and thelike). Unless stated otherwise specifically in the specification, analkyletherphopsho group may be optionally substituted. For example, incertain embodiments, the —Oalkyl or —Oalkylether moiety (R_(b)) in analkyletherphospho group is optionally substituted with one or more ofhydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether.“Oalkyletherphospho is an alkyletherphospho group connected to theremainder of the molecule via an oxygen atom. Unless stated otherwisespecifically in the specification, an Oalkyletherphospho group may beoptionally substituted.

“Alkylthiophospho” refers to the —P(═R_(a))(R_(b))R_(c) group, whereinR_(a) is O or S, R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d); and R_(c) is—Oalkyl or —Oalkylether, wherein R_(d) is a counter ion (e.g., Na+ andthe like) and provided that: R_(a) is S or R_(b) is S⁻ or SR_(d); orprovided that R_(a) is S and R_(b) is S⁻ or SR_(d). Unless statedotherwise specifically in the specification, a alkylthiophospho groupmay be optionally substituted. For example, in certain embodiments, the—Oalkyl or —Oalkylether moiety in a alkythiophospho group is optionallysubstituted with one or more of hydroxyl, amino, sulfhydryl, phosphate,thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether. “Oalkylthiophospho is a alkylthiophospho groupconnected to the remainder of the molecule via an oxygen atom. Unlessstated otherwise specifically in the specification, an Oalkylthiophosphogroup may be optionally substituted.

“Alkyletherthiophospho” refers to the —P(═R_(a))(R_(b))R_(c) group,wherein R_(a) is O or S, R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d); andR_(c) is —Oalkyl or —Oalkylether, wherein R_(d) is a counter ion (e.g.,Na+ and the like) and provided that: R_(a) is S or R_(b) is S⁻ orSR_(d); or provided that R_(a) is S and R_(b) is S⁻ or SR_(d). Unlessstated otherwise specifically in the specification, analkyletherthiophospho group may be optionally substituted. For example,in certain embodiments, the —Oalkyl or —Oalkylether moiety in aalkyletherthiophospho group is optionally substituted with one or moreof hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether.“Oalkyletherthiophospho is an alkyletherthiophospho group connected tothe remainder of the molecule via an oxygen atom. Unless statedotherwise specifically in the specification, an Oalkyletherthiophosphogroup may be optionally substituted.

“Amide” refers to the —NR_(a)R_(b) radical, wherein R_(a) and R_(b) areindependently H, alkyl or aryl. Unless stated otherwise specifically inthe specification, an amide group may be optionally substituted.

“Aryl” refers to a hydrocarbon ring system group comprising hydrogen, 6to 18 carbon atoms and at least one aromatic ring. For purposes of thisinvention, the aryl group may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems. Aryl groups include, but are not limited to, aryl groupsderived from aceanthrylene, acenaphthylene, acephenanthrylene,anthracene, azulene, benzene, chrysene, fluoranthene, fluorene,as-indacene, s-indacene, indane, indene, naphthalene, phenalene,phenanthrene, pleiadene, pyrene, and triphenylene. Unless statedotherwise specifically in the specification, the term “aryl” or theprefix “ar-” (such as in “aralkyl”) is meant to include aryl groups thatare optionally substituted.

“Aryloxy” refers to a group of the formula —OR_(a), where R_(a) is anaryl moiety as defined above, for example phenoxy and the like. Unlessstated otherwise specifically in the specification, an aryloxy group maybe optionally substituted.

“Aralkyl” refers to a group of the formula —R_(b)—R_(c) where R_(b) isan alkylene chain as defined above and R_(c) is one or more aryl groupsas defined above, for example, benzyl, diphenylmethyl and the like.Unless stated otherwise specifically in the specification, an aralkylgroup may be optionally substituted.

“Oaralkyl” is an aralkyl group which is connected to the remainder ofthe molecule via an oxygen linkage. “ODMT” refers to dimethoxytrityllinked to the rest of the molecule via an O atom. Unless statedotherwise specifically in the specification, an Oaralkyl group may beoptionally substituted.

“Cyanoalkyl” refers to an alkyl group comprising at least one cyanosubstituent. The one or more —CN substituents may be on a primary,secondary or tertiary carbon atom. Unless stated otherwise specificallyin the specification, a cyanoalkyl group may be optionally substituted.

“Cycloalkyl” or “carbocyclic ring” refers to a stable non-aromaticmonocyclic or polycyclic hydrocarbon group consisting solely of carbonand hydrogen atoms, which may include fused or bridged ring systems,having from three to fifteen carbon atoms, preferably having from threeto ten carbon atoms, and which is saturated or unsaturated and attachedto the rest of the molecule by a single bond. Monocyclic cycloalkylgroups include, for example, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl groupsinclude, for example, adamantyl, norbornyl, decalinyl,7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwisestated specifically in the specification, a cycloalkyl group may beoptionally substituted.

“Cycloalkylalkyl” refers to a group of the formula —R_(b)R_(d) whereR_(b) is an alkylene chain as defined above and R_(d) is a cycloalkylgroup as defined above. Unless stated otherwise specifically in thespecification, a cycloalkylalkyl group may be optionally substituted.

“Multicyclic” refers to any molecule having more than one ring. Therings may be either, fused, spirocyclic or separated by one or moreatoms (e.g., linked via an acyclic linker).

“Spirocyclic” refers to a multicyclic molecule wherein two rings share asingle carbon atom.

“Fused” refers to any ring structure described herein which is fused toan existing ring structure in the compounds of the invention. When thefused ring is a heterocyclyl ring or a heteroaryl ring, any carbon atomon the existing ring structure which becomes part of the fusedheterocyclyl ring or the fused heteroaryl ring may be replaced with anitrogen atom.

“Halo” or “halogen” refers to bromo, chloro, fluoro or iodo.

“Haloalkyl” refers to an alkyl group, as defined above, that issubstituted by one or more halo groups, as defined above, e.g.,trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl,1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and thelike. Unless stated otherwise specifically in the specification, ahaloalkyl group may be optionally substituted.

“Heterocyclyl” or “heterocyclic ring” refers to a stable 3- to18-membered non-aromatic ring group which consists of two to twelvecarbon atoms and from one to six heteroatoms selected from the groupconsisting of nitrogen, oxygen and sulfur. Unless stated otherwisespecifically in the specification, the heterocyclyl group may be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which mayinclude fused or bridged ring systems; and the nitrogen, carbon orsulfur atoms in the heterocyclyl group may be optionally oxidized; thenitrogen atom may be optionally quaternized; and the heterocyclyl groupmay be partially or fully saturated. Examples of such heterocyclylgroups include, but are not limited to, dioxolanyl,thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl,imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl,octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in thespecification, Unless stated otherwise specifically in thespecification, a heterocyclyl group may be optionally substituted.

“N-heterocyclyl” refers to a heterocyclyl group as defined abovecontaining at least one nitrogen and where the point of attachment ofthe heterocyclyl group to the rest of the molecule is through a nitrogenatom in the heterocyclyl group. Unless stated otherwise specifically inthe specification, a N-heterocyclyl group may be optionally substituted.

“Heterocyclylalkyl” refers to a group of the formula —R_(b)R_(e) whereR_(b) is an alkylene chain as defined above and R_(e) is a heterocyclylgroup as defined above, and if the heterocyclyl is a nitrogen-containingheterocyclyl, the heterocyclyl may be attached to the alkyl group at thenitrogen atom. Unless stated otherwise specifically in thespecification, a heterocyclylalkyl group may be optionally substituted.

“Heteroaryl” refers to a 5- to 14-membered ring system group comprisinghydrogen atoms, one to thirteen carbon atoms, one to six heteroatomsselected from the group consisting of nitrogen, oxygen and sulfur, andat least one aromatic ring. For purposes of this invention, theheteroaryl group may be a monocyclic, bicyclic, tricyclic or tetracyclicring system, which may include fused or bridged ring systems; and thenitrogen, carbon or sulfur atoms in the heteroaryl group may beoptionally oxidized; the nitrogen atom may be optionally quaternized.Examples include, but are not limited to, azepinyl, acridinyl,benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl,benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl,carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl,furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl,isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl,isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl,oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl,1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl,phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl,pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl,quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl,tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl,triazinyl, and thiophenyl (i.e., thienyl). Unless stated otherwisespecifically in the specification, a heteroaryl group may be optionallysubstituted.

“N-heteroaryl” refers to a heteroaryl group as defined above containingat least one nitrogen and where the point of attachment of theheteroaryl group to the rest of the molecule is through a nitrogen atomin the heteroaryl group. Unless stated otherwise specifically in thespecification, an N-heteroaryl group may be optionally substituted.

“Heteroarylalkyl” refers to a group of the formula —R_(b)R_(f) whereR_(b) is an alkylene chain as defined above and R_(f) is a heteroarylgroup as defined above. Unless stated otherwise specifically in thespecification, a heteroarylalkyl group may be optionally substituted.

“Hydroxylalkyl” refers to an alkyl group comprising at least onehydroxyl substituent. The one or more —OH substituents may be on aprimary, secondary or tertiary carbon atom. Unless stated otherwisespecifically in the specification, hydroxyalkyl group may be optionallysubstituted.

“Hydroxylalkylether” refers to an alkylether group comprising at leastone hydroxyl substituent. The one or more —OH substituents may be on aprimary, secondary or tertiary carbon atom. Unless stated otherwisespecifically in the specification, hydroxyalkylether group may beoptionally substituted.

“Phosphate” refers to the —OP(═O)(R_(a))R_(b) group, wherein R_(a) isOH, O⁻ or OR_(c); and R_(b) is OH, O⁻, OR_(c), a further phosphate group(as in diphosphate and triphosphate) thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether, whereinR_(c) is a counter ion (e.g., Na+ and the like). Unless stated otherwisespecifically in the specification, a phosphate group may be optionallysubstituted.

“Phospho” refers to the —P(═O)(R_(a))R_(b) group, wherein R_(a) is OH,O⁻ or OR_(c); and R_(b) is OH, O⁻, OR_(c), a phosphate group (as indiphosphate and triphosphate) thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether, whereinR_(c) is a counter ion (e.g., Na+ and the like). Unless stated otherwisespecifically in the specification, a phospho group may be optionallysubstituted.

“Phosphoalkyl” refers to the —P(═O)(R_(a))R_(b) group, wherein R_(a) isOH, O⁻ or OR_(c); and R_(b) is —Oalkyl, wherein R_(c) is a counter ion(e.g., Na+ and the like). Unless stated otherwise specifically in thespecification, a phosphoalkyl group may be optionally substituted. Forexample, in certain embodiments, the —Oalkyl moiety in a phosphoalkylgroup is optionally substituted with one or more of hydroxyl, amino,sulfhydryl or a phosphate, thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether.“Ophosphoalkyl is a phosphoalkyl group connected to the remainder of themolecule via an oxygen atom. Unless stated otherwise specifically in thespecification, an ophosphoalkyl group may be optionally substituted.

“Phosphoalkylether” refers to the —P(═O)(R_(a))R_(b) group, whereinR_(a) is OH, O⁻ or OR_(c); and R_(b) is —Oalkylether, wherein R_(c) is acounter ion (e.g., Na+ and the like). Unless stated otherwisespecifically in the specification, a phosphoalkylether group may beoptionally substituted. For example, in certain embodiments, the—Oalkylether moiety in a phosphoalkylether group is optionallysubstituted with one or more of hydroxyl, amino, sulfhydryl, phosphate,thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether. “Ophosphoalkylether is a phosphoalkylether groupconnected to the remainder of the molecule via an oxygen atom. Unlessstated otherwise specifically in the specification, anophosphoalkylether group may be optionally substituted.

“Sulfhydrylalkyl” refers to an alkyl group comprising at least onesulfhydryl substituent. The one or more —SH substituents may be on aprimary, secondary or tertiary carbon atom. Unless stated otherwisespecifically in the specification, a sulfhydrylalkyl group may beoptionally substituted.

“Sulfhydrylalkylether” refers to an alkylether group comprising at leastone sulfhydryl substituent. The one or more —SH substituents may be on aprimary, secondary or tertiary carbon atom. Unless stated otherwisespecifically in the specification, a sulfhydrylalkylether group may beoptionally substituted.

“Sulfonate” refers to the —OS(O)₂R_(a) group, wherein R_(a) is alkyl oraryl. Unless stated otherwise specifically in the specification, asulfonate group may be optionally substituted.

“Thioalkyl” refers to a group of the formula —SR_(a) where R_(a) is analkyl group as defined above containing one to twelve carbon atoms.Unless stated otherwise specifically in the specification, a thioalkylgroup may be optionally substituted.

“Thiophosphate” refers to the —OP(═R_(a))(R_(b))R_(c) group, whereinR_(a) is O or S, R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d); and R_(c) is OH,O⁻, OR_(d), phosphate group thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether, whereinR_(d) is a counter ion (e.g., Na+ and the like) and provided that: R_(a)is S or R_(b) is S⁻ or SR_(d); or provided that R_(a) is S and R_(b) isS⁻ or SR_(d). Unless stated otherwise specifically in the specification,a thiophosphate group may be optionally substituted.

“Thiophospho” refers to the —OP(═R_(a))(R_(b))R_(c) group, wherein R_(a)is O or S, R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d); and R_(c) is OH, O⁻,OR_(d), a phosphate group, thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether, whereinR_(d) is a counter ion (e.g., Na+ and the like) and provided that: R_(a)is S or R_(b) is S⁻ or SR_(d); or provided that R_(a) is S and R_(b) isS⁻ or SR_(d). Unless stated otherwise specifically in the specification,a thiophospho group may be optionally substituted.

“Thiophosphoalkyl” refers to the —P(═R_(a))(R_(b))R_(c) group, whereinR_(a) is O or S, R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d); and R_(c) is—Oalkyl, wherein R_(d) is a counter ion (e.g., Na+ and the like) andprovided that: R_(a) is S or R_(b) is S⁻ or SR_(d); or provided thatR_(a) is S and R_(b) is S⁻ or SR_(d). Unless stated otherwisespecifically in the specification, a thiophosphoalkyl group may beoptionally substituted. For example, in certain embodiments, the -Oalkylmoiety in a thiophosphoalkyl group is optionally substituted with one ormore of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate,phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether. “Othiophosphoalkyl is a thiophosphoalkyl groupconnected to the remainder of the molecule via an oxygen atom. Unlessstated otherwise specifically in the specification, an Othiophosphoalkylgroup may be optionally substituted.

“Thiophosphoalkylether” refers to the —P(═R_(a))(R_(b))R_(c) group,wherein R_(a) is O or S, R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d); andR_(c) is —Oalkylether, wherein R_(d) is a counter ion (e.g., Na+ and thelike) and provided that: R_(a) is S or R_(b) is S⁻ or SR_(d); orprovided that R_(a) is S and R_(b) is S⁻ or SR_(d). Unless statedotherwise specifically in the specification, a thiophosphoalkylethergroup may be optionally substituted. For example, in certainembodiments, the —Oalkylether moiety in a thiophosphoalkyl group isoptionally substituted with one or more of hydroxyl, amino, sulfhydryl,phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl,phosphoalkylether or thiophosphoalkylether. “Othiophosphoalkylether is athiophosphoalkylether group connected to the remainder of the moleculevia an oxygen atom. Unless stated otherwise specifically in thespecification, an Othiophosphoalkylether group may be optionallysubstituted.

The term “substituted” used herein means any of the above groups (i.e.,alkyl, alkylene, alkoxy, alkylamino, alkylether, alkylenether, amide,thioalkyl, aryl, aryloxy, aralkyl, Oaralkyl, cyanoalkyl, cycloalkyl,cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl,heterocyclylalkyl, heteroaryl, N-heteroaryl, heteroarylalkyl,hydroxylalkyl, aminoalkyl, hydroxylalkylether, phosphate, phosphoalkyl,phosphoalkylether, sulfhydrylalkyl, sulfhydrylalkylether, sulfonate,thiophosphate, thiophosphoalkyl and/or thiophosphoalkylether) wherein atleast one hydrogen atom is replaced by a bond to a non-hydrogen atomssuch as, but not limited to: a halogen atom such as F, Cl, Br, and I; anoxygen atom in groups such as hydroxyl groups, alkoxy groups, and estergroups; a sulfur atom in groups such as thiol groups, thioalkyl groups,sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atomin groups such as amines, amides, alkylamines, dialkylamines,arylamines, alkylarylamines, diarylamines, N-oxides, imides, andenamines; a silicon atom in groups such as trialkylsilyl groups,dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilylgroups; and other heteroatoms in various other groups. “Substituted”also means any of the above groups in which one or more hydrogen atomsare replaced by a higher-order bond (e.g., a double- or triple-bond) toa heteroatom such as oxygen in oxo, carbonyl, carboxyl, and estergroups; and nitrogen in groups such as imines, oximes, hydrazones, andnitriles. For example, “substituted” includes any of the above groups inwhich one or more hydrogen atoms are replaced with —NR_(g)R_(h),—NR_(g)C(═O)R_(h), —NR_(g)C(═O)NR_(g)R_(h), —NR_(g)C(═O)OR_(h),—NR_(g)SO₂R_(h), —OC(═O)NR_(g)R_(h), —OR_(g), —SR_(g), —SOR_(g),—SO₂R_(g), —OSO₂R_(g), —SO₂OR_(g), ═NSO₂R_(g), and —SO₂NR_(g)R_(h).“Substituted also means any of the above groups in which one or morehydrogen atoms are replaced with —C(═O)R_(g), —C(═O)OR_(g),—C(═O)NR_(g)R_(h), —CH₂SO₂R_(g), —CH₂SO₂NR_(g)R_(h). In the foregoing,R_(g) and R_(h) are the same or different and independently hydrogen,alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl,cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl,heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl.“Substituted” further means any of the above groups in which one or morehydrogen atoms are replaced by a bond to an amino, cyano, hydroxyl,imino, nitro, oxo, thioxo, halo, alkyl, alkoxy, alkylamino, thioalkyl,aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl,N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/orheteroarylalkyl group. In addition, each of the foregoing substituentsmay also be optionally substituted with one or more of the abovesubstituents.

“Conjugation” refers to the overlap of one p-orbital with anotherp-orbital across an intervening sigma bond. Conjugation may occur incyclic or acyclic compounds. A “degree of conjugation” refers to theoverlap of at least one p-orbital with another p-orbital across anintervening double bond. For example, 1,3-butadine has one degree ofconjugation, while benzene and other aromatic compounds typically havemultiple degrees of conjugation. Fluorescent and colored compoundstypically comprise at least one degree of conjugation.

“Fluorescent” refers to a molecule which is capable of absorbing lightof a particular frequency and emitting light of a different frequency.Fluorescence is well-known to those of ordinary skill in the art.

“Colored” refers to a molecule which absorbs light within the coloredspectrum (i.e., red, yellow, blue and the like).

A “linker” refers to a contiguous chain of at least one atom, such ascarbon, oxygen, nitrogen, sulfur, phosphorous and combinations thereof,which connects a portion of a molecule to another portion of the samemolecule or to a different molecule, moiety or solid support (e.g.,microparticle). Linkers may connect the molecule via a covalent bond orother means, such as ionic or hydrogen bond interactions.

For purposes of the present invention, the term “biomolecule” refers toany of a variety of biological materials, including nucleic acids,carbohydrates, amino acids, polypeptides, glycoproteins, hormones,aptamers and mixtures thereof. More specifically, the term is intendedto include, without limitation, RNA, DNA, oligonucleotides, modified orderivatized nucleotides, enzymes, receptors, prions, receptor ligands(including hormones), antibodies, antigens, and toxins, as well asbacteria, viruses, blood cells, and tissue cells. The visuallydetectable biomolecules of the invention (i.e., compounds of structure(I) having a biomolecule linked thereto) are prepared, as furtherdescribed herein, by contacting a biomolecule with a compound having areactive group that enables attachment of the biomolecule to thecompound via any available atom or functional group, such as an amino,hydroxy, carboxyl, or sulfhydryl group on the biomolecule.

The terms “visible” and “visually detectable” are used herein to referto substances that are observable by visual inspection, without priorillumination, or chemical or enzymatic activation. Such visuallydetectable substances absorb and emit light in a region of the spectrumranging from about 300 to about 900 nm. Preferably, such substances areintensely colored, preferably having a molar extinction coefficient ofat least about 40,000, more preferably at least about 50,000, still morepreferably at least about 60,000, yet still more preferably at leastabout 70,000, and most preferably at least about 80,000 M⁻¹cm⁻¹. Thebiomolecules of the invention may be detected by observation with thenaked eye, or with the aid of a optically based detection device,including, without limitation, absorption spectrophotometers,transmission light microscopes, digital cameras and scanners. Visuallydetectable substances are not limited to those which emit and/or absorblight in the visible spectrum. Substances which emit and/or absorb lightin the ultraviolet (UV) region (about 10 nm to about 400 nm), infrared(IR) region (about 700 nm to about 1 mm), and substances emitting and/orabsorbing in other regions of the electromagnetic spectrum are alsoincluded with the scope of “visually detectable” substances.

For purposes of the invention, the term “photostable visible dye” refersto a chemical moiety that is visually detectable, as definedhereinabove, and is not significantly altered or decomposed uponexposure to light. Preferably, the photostable visible dye does notexhibit significant bleaching or decomposition after being exposed tolight for at least one hour. More preferably, the visible dye is stableafter exposure to light for at least 12 hours, still more preferably atleast 24 hours, still yet more preferably at least one week, and mostpreferably at least one month. Nonlimiting examples of photostablevisible dyes suitable for use in the compounds and methods of theinvention include azo dyes, thioindigo dyes, quinacridone pigments,dioxazine, phthalocyanine, perinone, diketopyrrolopyrrole,quinophthalone, and truarycarbonium.

As used herein, the term “perylene derivative” is intended to includeany substituted perylene that is visually detectable. However, the termis not intended to include perylene itself. The terms “anthracenederivative”, “naphthalene derivative”, and “pyrene derivative” are usedanalogously. In some preferred embodiments, a derivative (e.g.,perylene, pyrene, anthracene or naphthalene derivative) is an imide,bisimide or hydrazamimide derivative of perylene, anthracene,naphthalene, or pyrene.

The visually detectable biomolecules of the invention are useful for awide variety of biochemical and biomedical applications in which thereis a need to determine the presence, location, or quantity of aparticular biomolecule. In another aspect, therefore, the inventionprovides a method for visually detecting a biomolecule, comprising: (a)providing a biological system with a visually detectable biomoleculecomprising the compound of structure (I) linked to a biomolecule; and(b) detecting the biomolecule by its visible properties. For purposes ofthe invention, the phrase “detecting the biomolecule by its visibleproperties” means that the biomolecule, without illumination or chemicalor enzymatic activation, is observed with the naked eye, or with the aidof a optically based detection device, including, without limitation,absorption spectrophotometers, transmission light microscopes, digitalcameras and scanners. A densitometer may be used to quantify the amountof visually detectable biomolecule present. For example, the relativequantity of the biomolecule in two samples can be determined bymeasuring relative optical density. If the stoichiometry of dyemolecules per biomolecule is known, and the extinction coefficient ofthe dye molecule is known, then the absolute concentration of thebiomolecule can also be determined from a measurement of opticaldensity. As used herein, the term “biological system” is used to referto any solution or mixture comprising one or more biomolecules inaddition to the visually detectable biomolecule. Nonlimiting examples ofsuch biological systems include cells, cell extracts, tissue samples,electrophoretic gels, assay mixtures, and hybridization reactionmixtures.

“Microparticle” refers to any of a number of small particles useful forattachment to compounds of the invention, including, but not limited to,glass beads, magnetic beads, polymeric beads, nonpolymeric beads, andthe like. In certain embodiments, a microparticle comprises polystyrenebeads.

“Base pairing moiety” refers to a heterocyclic moiety capable ofhybridizing with a complementary heterocyclic moiety via hydrogen bonds(e.g., Watson-Crick base pairing). Base pairing moieties include naturaland unnatural bases. Non-limiting examples of base pairing moieties areRNA and DNA bases such adenosine, guanosine, thymidine, cytosine anduridine and analogues thereof.

The invention disclosed herein is also meant to encompass all compoundsof structure (I) being isotopically-labelled by having one or more atomsreplaced by an atom having a different atomic mass or mass number.Examples of isotopes that can be incorporated into the disclosedcompounds include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine, chlorine, and iodine, such as ²H, ³H, ¹¹C, ¹³C,¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I and ¹²⁵I,respectively.

Isotopically-labeled compounds of structure (I) can generally beprepared by conventional techniques known to those skilled in the art orby processes analogous to those described below and in the followingExamples using an appropriate isotopically-labeled reagent in place ofthe non-labeled reagent previously employed.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

“Optional” or “optionally” means that the subsequently described eventor circumstances may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. For example, “optionally substituted alkyl” means that thealkyl group may or may not be substituted and that the descriptionincludes both substituted alkyl groups and alkyl groups having nosubstitution.

“Salt” includes both acid and base addition salts.

“Acid addition salt” refers to those salts which are formed withinorganic acids such as, but not limited to, hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, and organic acids such as, but not limited to, acetic acid,2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid,aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoicacid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproicacid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamicacid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonicacid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid,galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid,glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid,glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid,lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid,malonic acid, mandelic acid, methanesulfonic acid, mucic acid,naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid,1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid,oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamicacid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid,stearic acid, succinic acid, tartaric acid, thiocyanic acid,p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and thelike.

“Base addition salt” refers to those salts which are prepared fromaddition of an inorganic base or an organic base to the free acid. Saltsderived from inorganic bases include, but are not limited to, sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Salts derived from organic basesinclude, but are not limited to, salts of primary, secondary, andtertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asammonia, isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

Often crystallizations produce a solvate of the compound of theinvention. The present invention includes all solvates of the describedcompounds. As used herein, the term “solvate” refers to an aggregatethat comprises one or more molecules of a compound of the invention withone or more molecules of solvent. The solvent may be water, in whichcase the solvate may be a hydrate. Alternatively, the solvent may be anorganic solvent. Thus, the compounds of the present invention may existas a hydrate, including a monohydrate, dihydrate, hemihydrate,sesquihydrate, trihydrate, tetrahydrate and the like, as well as thecorresponding solvated forms. The compounds of the invention may be truesolvates, while in other cases the compounds of the invention may merelyretain adventitious water or another solvent or be a mixture of waterplus some adventitious solvent.

The compounds of the invention, or their salts, tautomers or solvatesmay contain one or more asymmetric centers and may thus give rise toenantiomers, diastereomers, and other stereoisomeric forms that may bedefined, in terms of absolute stereochemistry, as (R)- or (S)- or, as(D)- or (L)- for amino acids. The present invention is meant to includeall such possible isomers, as well as their racemic and optically pureforms. Optically active (+) and (−), (R)- and (S)-, or (D)- and(L)-isomers may be prepared using chiral synthons or chiral reagents, orresolved using conventional techniques, for example, chromatography andfractional crystallization. Conventional techniques for thepreparation/isolation of individual enantiomers include chiral synthesisfrom a suitable optically pure precursor or resolution of the racemate(or the racemate of a salt or derivative) using, for example, chiralhigh pressure liquid chromatography (HPLC). When the compounds describedherein contain olefinic double bonds or other centers of geometricasymmetry, and unless specified otherwise, it is intended that thecompounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present invention contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers”,which refers to two stereoisomers whose molecules are nonsuperimposeablemirror images of one another.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. The present invention includestautomers of any said compounds. Various tautomeric forms of thecompounds are easily derivable by those of ordinary skill in the art.

The chemical naming protocol and structure diagrams used herein are amodified form of the I.U.P.A.C. nomenclature system, using the ACD/NameVersion 9.07 software program and/or ChemDraw Version 10.0 softwarenaming program (CambridgeSoft). Common names familiar to one of ordinaryskill in the art are also used.

As noted above, in one embodiment of the present invention, compoundsuseful as fluorescent and/or colored dyes in various analytical methodsare provided. The compounds have the following structure (I):

or a stereoisomer, tautomer or salt thereof, wherein:

M¹ and M² are, at each occurrence, independently a moiety comprising twoor more double bonds and at least one degree of conjugation, and atleast one occurrence of M¹ is a moiety comprising three or more aryl orheteroaryl rings, or combinations thereof;

A represents a cyclic moiety;

L¹, L², L³, L⁴, L⁵ and L⁶ are, at each occurrence, independentlyoptional linkers comprising atoms selected from carbon, oxygen, sulfur,nitrogen and phosphorous;

R¹ is, at each occurrence, independently H, alkyl or alkoxy;

R² and R³ are independently H, OH, SH, —NH₂, alkyl, alkylether,hydroxylalkyl, aminoalkyl, hydroxylalkylether, sulfhydrylalkyl,sulfyhdrylalkylether, cyanoalkyl, —Oaralkyl, phosphate, thiophosphate,phospho, thiophospho, alkylphospho, alkylthiophospho, —Oalkylphospho,—Oalkylthiophospho, alkyletherphospho, alkyletherthiophospho,—Oalkyletherphospho, —Oalkyletherthiophospho phosphoalkyl,phosphoalkylether, thiophosphoalkyl, thiophosphoalkylether,—Ophosphoalkyl, O-phosphoalkylether, —Othiophosphoalkyl or—Othiophosphoalkylether, or R² is a linker comprising a covalent bond toa biomolecule or microparticle, and R³ is H, OH, SH, alkyl, alkylether,hydroxylalkyl, aminoalkyl, hydroxylalkylether, sulfhydrylalkyl,sulfyhdrylalkylether, cyanoalkyl, —Oaralkyl, phosphate, thiophosphate,alkylphospho, alkylthiophospho, —Oalkylphospho, —Oalkylthiophospho,alkyletherphospho, alkyletherthiophospho, —Oalkyletherphospho,—Oalkyletherthiophospho phosphoalkyl, phosphoalkylether,thiophosphoalkyl, thiophosphoalkylether, —Ophosphoalkyl,O-phosphoalkylether, —Othiophosphoalkyl or —Othiophosphoalkylether,;

n is an integer from 1 to 20; and

q and w are each independently 0 or 1 for each integral value of n,wherein q is 1 for at least one integral value of n.

In some embodiments, n is an integer from 1 to 10 or from 2 to 10.

In certain embodiments, L² comprises phosphorous-oxygen bonds. In otherembodiments, L⁵ comprises phosphorous-oxygen bonds.

For example, in some embodiments the compound has the followingstructure (III):

wherein:

M¹ and M² are, at each occurrence, independently a moiety comprising twoor more double bonds and at least one degree of conjugation, and atleast one occurrence of M¹ is a moiety comprising three or more aryl orheteroaryl rings, or combinations thereof;

L¹, L³, L⁴ L⁶, L⁷ and L⁸ are, at each occurrence, independently optionalalkylene or heteroalkylene linkers;

R¹ is, at each occurrence, independently H, alkyl or alkoxy;

R² is an electron pair, H, alkyl, alkylether, hydroxylalkyl, aminoalkyl,hydroxylalkylether, sulfhydrylalkyl, sulfyhdrylalkylether, cyanoalkyl,phospho, thiophospho, alkylphospho, alkylthiophospho, alkyletherphospho,alkyletherthiophospho, phosphoalkyl, phosphoalkylether, thiophosphoalkylor thiophosphoalkylether, or R² is a linker comprising a covalent bondto a biomolecule or microparticle;

R³ is H, OH, SH, —NH₂, alkyl, alkylether, hydroxylalkyl, aminoalkyl,hydroxylalkylether, sulfhydrylalkyl, sulfyhdrylalkylether, cyanoalkyl,—Oaralkyl, phosphate, thiophosphate, alkylphospho, alkylthiophospho,—Oalkylphospho, —Oalkylthiophospho, alkyletherphospho,alkyletherthiophospho, —Oalkyletherphospho, —Oalkyletherthiophosphophosphoalkyl, phosphoalkylether, thiophosphoalkyl,thiophosphoalkylether, —Ophosphoalkyl, O-phosphoalkylether,—Othiophosphoalkyl or —Othiophosphoalkylether;

R⁴ is, at each occurrence, independently O⁻, S⁻, OZ, SZ or N(R⁶)₂, whereZ is a cation and each R⁶ is independently H or alkyl;

R⁵ is, at each occurrence, independently oxo, thioxo or absent;

n is an integer from 1 to 20; and

q and w are each independently 0 or 1 for each integral value of n,wherein q is 1 for at least one integral value of n.

In some embodiments, the compound of structure (III) comprises at leasttwo occurrences of the “q” unit or at least one occurrence of the “q”unit and at least one occurrence of the “w” unit. For clarity, the “q”and “w” units have the following structures:

Accordingly, in some embodiments of structure (III):

n is an integer from 2 to 20, and

q and w are each independently 0 or 1 for each integral value of n,wherein q is 1 for at least two integral values of n, or wherein q and ware each independently one for at least one integral value of n, whichintegral value may be the same or different.

In any of the foregoing embodiments, n is an integer from 2-15 or n is2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

In some embodiments of compounds (I) or (III), w is 0 for each integralvalue of n. For example, in some embodiments the compound has thefollowing structure (II):

or a stereoisomer, tautomer or salt thereof, wherein:

M¹ is, at each occurrence, independently a moiety comprising two or moredouble bonds and at least one degree of conjugation, and at least oneoccurrence of M¹ is a moiety comprising three or more aryl or heteroarylrings, or combinations thereof;

L¹, L² and L³ are, at each occurrence, independently optional linkerscomprising atoms selected from carbon, oxygen, sulfur, nitrogen andphosphorous;

R¹ is, at each occurrence, independently H, alkyl or alkoxy;

R² and R³ are independently H, OH, SH, —NH₂, alkyl, alkylether,hydroxylalkyl, aminoalkyl, hydroxylalkylether, sulfhydrylalkyl,sulfyhdrylalkylether, cyanoalkyl, —Oaralkyl, phosphate, thiophosphate,phospho, thiophospho, alkylphospho, alkylthiophospho, —Oalkylphospho,—Oalkylthiophospho, alkyletherphospho, alkyletherthiophospho,—Oalkyletherphospho, —Oalkyletherthiophospho phosphoalkyl,phosphoalkylether, thiophosphoalkyl, thiophosphoalkylether,—Ophosphoalkyl, O-phosphoalkylether, —Othiophosphoalkyl or—Othiophosphoalkylether, or R² is a linker comprising a covalent bond toa biomolecule or microparticle, and R³ is H, OH, SH, alkyl, alkylether,hydroxylalkyl, aminoalkyl, hydroxylalkylether, sulfhydrylalkyl,sulfyhdrylalkylether, cyanoalkyl, —Oaralkyl, phosphate, thiophosphate,alkylphospho, alkylthiophospho, —Oalkylphospho, —Oalkylthiophospho,alkyletherphospho, alkyletherthiophospho, —Oalkyletherphospho,—Oalkyletherthiophospho phosphoalkyl, phosphoalkylether,thiophosphoalkyl, thiophosphoalkylether, —Ophosphoalkyl,O-phosphoalkylether, —Othiophosphoalkyl or —Othiophosphoalkylether; and

n is an integer from 1 to 20, for example from 2 to 20 or from 2 to 10.

In other embodiments of the foregoing, w is 0 for each integral value ofn, and the compound has the following structure (IIa):

wherein:

M¹ is, at each occurrence, independently a moiety comprising two or moredouble bonds and at least one degree of conjugation, and at least oneoccurrence of M¹ is a moiety comprising four or more aryl or heteroarylrings, or combinations thereof;

L¹, L⁷ and L³ are, at each occurrence, independently optional alkyleneor heteroalkylene linkers;

R¹ is, at each occurrence, independently H, alkyl or alkoxy;

R² is H, alkyl, alkylether, hydroxylalkyl, aminoalkyl,hydroxylalkylether, sulfhydrylalkyl, sulfyhdrylalkylether, cyanoalkyl,phospho, thiophospho, alkylphospho, alkylthiophospho, alkyletherphospho,alkyletherthiophospho, phosphoalkyl, phosphoalkylether, thiophosphoalkylor thiophosphoalkylether, or R² is a linker comprising a covalent bondto a biomolecule or microparticle;

R³ is H, OH, SH, —NH₂, alkyl, alkylether, hydroxylalkyl, aminoalkyl,hydroxylalkylether, sulfhydrylalkyl, sulfyhdrylalkylether, cyanoalkyl,—Oaralkyl, phosphate, thiophosphate, alkylphospho, alkylthiophospho,—Oalkylphospho, —Oalkylthiophospho, alkyletherphospho,alkyletherthiophospho, —Oalkyletherphospho, —Oalkyletherthiophosphophosphoalkyl, phosphoalkylether, thiophosphoalkyl,thiophosphoalkylether, —Ophosphoalkyl, O-phosphoalkylether,—Othiophosphoalkyl or —Othiophosphoalkylether;

R⁴ is, at each occurrence, independently O⁻, S⁻, OZ, SZ or N(R⁶)₂, whereZ is a cation and each R⁶ is independently H or alkyl;

R⁵ is, at each occurrence, independently oxo, thioxo or absent; and

n is an integer from 1 to 20, for example from 2 to 20 or from 2 to 10.

In still more embodiments, the compound has the following structure(IIb):

wherein:

R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are, at each occurrence, independently H oralkyl; and

x¹, y¹ and z¹ are, at each occurrence, independently an integer from 0to 5.

In other embodiments, the compound has one of the following structures(IIc), (IId), (IIe) or (IIf):

In some embodiments of compounds (IIb), (IIc), (IId), (IIe) or (IIf),x¹, y¹ and z¹ are, at each occurrence, 1. In other embodiments ofcompounds (IIb), (IIc), (IId), (IIe) or (IIf), x¹ is 0, at eachoccurrence, and y¹ and z¹ are, at each occurrence, 1. In otherembodiments of compounds (IIb), (IIc), (IId), (IIe) or (IIf), n is aninteger from 2 to 10. For example, in some embodiments n is 2, 3, 4, 5,6, 7, 8, 9, or 10.

In still more embodiments, w is 1 for at least one integral value of n.For example, in some embodiments, the compound has the followingstructure (IIIa):

or a stereoisomer, tautomer or salt thereof, wherein:

M¹ and M² are, at each occurrence, independently a moiety comprising twoor more double bonds and at least one degree of conjugation;

L¹, L², L³, L⁴, L⁵ and L⁶ are, at each occurrence, independentlyoptional linkers comprising atoms selected from carbon, oxygen, sulfur,nitrogen and phosphorous;

R¹ is, at each occurrence, independently H, alkyl or alkoxy;

R² and R³ are independently H, OH, SH, —NH₂, alkyl, alkylether,hydroxylalkyl, aminoalkyl, hydroxylalkylether, sulfhydrylalkyl,sulfyhdrylalkylether, cyanoalkyl, —Oaralkyl, phosphate, thiophosphate,phospho, thiophospho, alkylphospho, alkylthiophospho, —Oalkylphospho,—Oalkylthiophospho, alkyletherphospho, alkyletherthiophospho,—Oalkyletherphospho, —Oalkyletherthiophospho phosphoalkyl,phosphoalkylether, thiophosphoalkyl, thiophosphoalkylether,—Ophosphoalkyl, O-phosphoalkylether, —Othiophosphoalkyl or—Othiophosphoalkylether, or R² is a linker comprising a covalent bond toa biomolecule or microparticle, and R³ is H, OH, SH, alkyl, alkylether,hydroxylalkyl, aminoalkyl, hydroxylalkylether, sulfhydrylalkyl,sulfyhdrylalkylether, cyanoalkyl, —Oaralkyl, phosphate, thiophosphate,alkylphospho, alkylthiophospho, —Oalkylphospho, —Oalkylthiophospho,alkyletherphospho, alkyletherthiophospho, —Oalkyletherphospho,—Oalkyletherthiophospho phosphoalkyl, phosphoalkylether,thiophosphoalkyl, thiophosphoalkylether, —Ophosphoalkyl,O-phosphoalkylether, —Othiophosphoalkyl or —Othiophosphoalkylether;

n is an integer from 1 to 10; and

q and w are each independently 0 or 1 for each integral value of n, andq and w are each 1 for at least one integral value of n.

In still other embodiments, the compound has the following structure(IIIb):

wherein:

R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are, at each occurrence, independently H oralkyl; and

x¹, x², y¹, y², z¹ and z² are, at each occurrence, independently aninteger from 0 to 5.

In some of the foregoing embodiments, R⁵ is oxo and R⁴ is O⁻ or OZ. Inother of the foregoing embodiments, R² is H or an electron pair.

In other of any of the foregoing, R² is hydroxylalkyl, aminoalkyl,hydroxylalkylether, sulfhydrylalkyl or sulfhydrylalkylether. Forexample, in some embodiments R² has one of the following structures:

wherein:

R^(2a) is —OH, —NH₂, or —SH; and

a is an integer from 1 to 10.

In still other of any of the foregoing, R² is alkylphospho,alkylthiophospho, alkyletherphospho, alkyletherthiophospho,phosphoalkyl, phosphoalkylether, thiophosphoalkyl orthiophosphoalkylether optionally substituted with a substituent selectedfrom —OH, —NH₂, and —SH. For example, in some embodiments R² has one ofthe following structures:

wherein:

R^(2a) is —OH, —SH, —NH₂, phosphate or thiophosphate;

R^(4a) and R^(4b) are independently O⁻, S⁻, OZ or SZ, where Z is acation;

R^(5a) and R^(5b) are independently oxo, or thioxo; and

a, b and c are each independently integers from 1 to 10.

In some embodiments, R³ is OH. In still other embodiments of any of theforegoing, R³ is phosphate, thiophosphate, phospho, thiophospho,—Oalkylphospho, —Oalkylthiophospho, —Oalkyletherphospho,—Oalkyletherthiophospho, —Ophosphoalkyl, —Ophosphoalkylether,—Othiophosphoalkyl or —Othiophosphoalkylether optionally substitutedwith a substituent selected from —OH. —NH₂, and —SH.

For example, in some embodiments R³ has one of the following structures:

wherein:

R^(3a) is —OH, —SH, —NH₂, phosphate or thiophosphate;

R^(4a) and R^(4b) are independently O⁻, S⁻, OZ or SZ, where Z is acation;

R^(5a) and R^(5b) are independently oxo, or thioxo; and

b and c are each independently integers from 1 to 10.

In still other of any of the foregoing, R² is alkylphospho,alkylthiophospho, alkyletherphospho, alkyletherthiophospho,phosphoalkyl, phosphoalkylether, thiophosphoalkyl orthiophosphoalkylether optionally substituted with a substituent selectedfrom —OH, —NH₂, and —SH and R³ is phosphate, thiophosphate, phospho,thiophospho, —Oalkylphospho, —Oalkylthiophospho, —Oalkyletherphospho,—Oalkyletherthiophospho, —Ophosphoalkyl, —Ophosphoalkylether,—Othiophosphoalkyl or —Othiophosphoalkylether optionally substitutedwith a substituent selected from —OH, —NH₂, and —SH.

In even more embodiments R² has one of the following structures:

wherein:

R^(2a) is —OH, —SH, —NH₂, phosphate or thiophosphate;

R^(4a) and R^(4b) are independently O⁻, S⁻, OZ or SZ, where Z is acation;

R^(5a) and R^(5b) are independently oxo, or thioxo; and

a, b and c are each independently integers from 1 to 10, and

R³ has one of the following structures:

wherein:

R^(3a) is —OH, —SH, —NH₂, phosphate or thiophosphate;

R^(4a) and R^(4b) are independently O⁻, S⁻, OZ or SZ, where Z is acation;

R^(5a) and R^(5b) are independently oxo, or thioxo; and

b and c are each independently integers from 1 to 10.

In other embodiments of the above, R^(4a) and R^(4b) are each O⁻ andR^(5a) and R^(5b) are each oxo. In some other embodiments, R^(4a) andR^(4b) are each O⁻ and R^(5a) and R^(5b) are each thioxo. In otherembodiments, R^(4a) and R^(4b) are each S⁻ and R^(5a) and R^(5b) areeach thioxo. In still other embodiments, R^(4a) and R^(4b) are each S⁻and R^(5a) and R^(5b) are each oxo.

In more embodiments of the foregoing, at least one of a, b or c is 2.For example, in some embodiments each of a, b and c is 2.

In some different embodiments, at least one of a, b or c is 6. Forexample, in some embodiments each of a, b and c is 6.

In some embodiments of any of the foregoing compounds of structure (I),(II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf) (III), (IIIa) or (IIIb),n is an integer from 1 to 5, from 2 to 15, from 2 to 10 or from 2 to 5.

In some embodiments of the above foregoing compounds of structure (I),(II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf) (III), (IIIa) or (IIIb),R^(2a) or R^(3a) (or both) are alkylphospho, alkylthiophospho,alkyletherphospho, alkyletherthiophospho, phosphoalkyl,phosphoalkylether, thiophosphoalkyl or thiophosphoalkylether optionallysubstituted with a substituent selected from —OH, —NH₂, and —SH. Forexample, in some embodiments of any of the foregoing, R² or R³ (or both:have the following structure:

In some embodiments of any the above foregoing compounds of structure(III), (IIIa) or (IIIb), R² is H or an electron pair, R³ is phosphateand the sum of q and w is at least 2. In some of these embodiments, q is2 or more, for example 3 or more. In other of these embodiments, each ofL¹, L³ and L⁷ are alkylene linkers, for example methylene. In still moreembodiments, L¹ and L³ are alkylene linkers, such as methylene, and L⁷is absent (i.e., a direct bond). In still more of the forgoingembodiments, M¹ comprises four or more aryl or heteroaryl rings, orcombinations thereof, for example five or more.

In some other embodiments of any the above foregoing compounds ofstructure (I), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf) (III),(IIIa) or (IIIb), L³ is a heteroalkylene linker, for example aheteroalkylene linker comprising O—P—O bonds, S—S bonds, or combinationsthereof. In some of these embodiments, R² is H or an electron pair.

In other embodiments, a compound having the following structure (IV) isprovided:

wherein:

M¹ is, at each occurrence, independently a moiety comprising two or moredouble bonds and at least one degree of conjugation, and at least oneoccurrence of M¹ is a moiety comprising three or more aryl or heteroarylrings, or combinations thereof;

L¹, L³, and L⁷ are, at each occurrence, independently optional alkyleneor heteroalkylene linkers;

R¹ is, at each occurrence, independently H, alkyl or alkoxy;

R² is phospho, thiophospho, alkylphospho, alkylthiophospho,alkyletherphospho, alkyletherthiophospho, phosphoalkyl,phosphoalkylether, thiophosphoalkyl or thiophosphoalkylether, or R² is alinker comprising a covalent bond to a biomolecule or microparticle;

R³ is H, OH, SH, —NH₂, alkyl, alkylether, hydroxylalkyl, aminoalkyl,hydroxylalkylether, sulfhydrylalkyl, sulfyhdrylalkylether, phosphate,thiophosphate, alkylphospho, alkylthiophospho, —Oalkylphospho,—Oalkylthiophospho, alkyletherphospho, alkyletherthiophospho,—Oalkyletherphospho, —Oalkyletherthiophospho phosphoalkyl,phosphoalkylether, thiophosphoalkyl, thiophosphoalkylether,—Ophosphoalkyl, O-phosphoalkylether, —Othiophosphoalkyl or—Othiophosphoalkylether;

R⁴ is, at each occurrence, independently O⁻, S⁻, OZ, SZ or N(R⁶)₂, whereZ is a cation and each R⁶ is independently H or alkyl;

R⁵ is, at each occurrence, independently oxo, thioxo or absent; and

n is an integer from 1 to 20, for example 1 to 10.

In some embodiments of compound (IV), R² is alkylphospho,alkylthiophospho, alkyletherphospho, alkyletherthiophospho,phosphoalkyl, phosphoalkylether, thiophosphoalkyl orthiophosphoalkylether, wherein R² is optionally substituted with asubstituent selected from —OH, —NH₂, and —SH.

For example, in certain embodiments of compound (IV), R² has one of thefollowing structures:

wherein:

R^(2a) is —OH, —SH, —NH₂, phosphate or thiophosphate;

R^(4a) and R^(4b) are independently O⁻, S⁻, OZ or SZ, where Z is acation;

R^(5a) and R^(5b) are independently oxo, or thioxo; and

a, b and c are each independently integers from 1 to 10.

In some embodiments of compound (IV), R³ is OH. In differentembodiments, R³ is, phosphate, thiophosphate, phospho, thiophospho,—Oalkylphospho, —Oalkylthiophospho, —Oalkyletherphospho,—Oalkyletherthiophospho, —Ophosphoalkyl, —Ophosphoalkylether,—Othiophosphoalkyl or —Othiophosphoalkylether optionally substitutedwith a substituent selected from —OH, —NH₂, and —SH.

In still other embodiments of compound (IV), R³ has one of the followingstructures:

wherein:

R^(3a) is —OH, —SH, —NH₂, phosphate or thiophosphate;

R^(4a) and R^(4b) are independently O⁻, S⁻, OZ or SZ, where Z is acation;

R^(5a) and R^(5b) are independently oxo, or thioxo; and

b and c are each independently integers from 1 to 10.

In some other embodiments of compound (IV), R⁴ is O⁻ and R⁵ is oxo ateach occurrence.

In other embodiments of compound (IV), L¹, L³ and L⁷ are each alkylenelinkers. In different embodiments, L¹ and L³ are each alkylene linkersand L⁷ is absent. In some of the foregoing embodiments, alkylene ismethylene.

In some of the foregoing embodiments, R³ is —OH. In other embodiments,R² is H (thus at certain pH values, the oxygen atom is negativelycharged, i.e., R² is an electron pair, since the H is acidic).

In some embodiments of structure (IV), L⁷ and L³ are, at eachoccurrence, independently optional alkylene, phosphoalkylene orphosphoalkylenether linkers. In some embodiments, L⁷ or L³ or both arepresent. In some embodiments, L⁷ and L³ are, at each occurrence,independently selected from:

wherein:

R^(2a) is —OH, —SH, —NH₂, phosphate or thiophosphate;

R^(4a) and R^(4b) are independently O⁻, S⁻, OZ or SZ, where Z is acation;

R^(5a) and R^(5b) are independently oxo, or thioxo; and

a, b and c are each independently integers from 1 to 10.

In some other embodiments of any the above foregoing compounds ofstructure (IV), one or more occurrences of L³ is a heteroalkylenelinker, for example a heteroalkylene linker comprising O—P—O bonds, S—Sbonds, or combinations thereof. In some of these embodiments, R² is H oran electron pair. For example, in some embodiments at least oneoccurrence of L³ has one of the following structures:

In still other embodiments, a compound having the following structure(Ig) is provided:

wherein:

M¹ is a moiety comprising three or more aryl or heteroaryl rings, orcombinations thereof;

R¹ is H, C₁-C₆ alkyl or alkoxy;

R² is cyanoalkyl;

R³ is H or —Oaralkyl;

R⁶ is C₁-C₆ alkyl;

R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are, at each occurrence, independently H oralkyl; and

x, y and z are, at each occurrence, independently an integer from 0 to5.

In some embodiments of the compound of structure (Ig), each R⁶ isisopropyl. In other embodiments, R² is 2-cyanoethyl. In still moreembodiments, R³ is —Oaralkyl, for example —O-dimethoxytrityl (—ODMT).

In different embodiments of compound (Ig), x, y and z are each 1. Inother embodiments, x is 0 and y and z are each 1.

In other embodiments, a compound having the following structure (Ih) isprovided:

wherein:

M¹ is a moiety comprising three or more aryl or heteroaryl rings, orcombinations thereof;

R² is H, an electron pair or a cation;

R³ is OH;

R⁴ is O⁻, S⁻, OZ, SZ where Z is a cation;

R⁵ is oxo or thioxo;

R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are, at each occurrence, independently H oralkyl; and

x, y and z are, at each occurrence, independently an integer from 0 to5.

In some embodiments of (Ih), x, y and z are each 1. In otherembodiments, x is 0 and y and z are each 1. In still more embodiments,R⁴ is O⁻ or OZ and R⁵ is oxo.

Other embodiments are directed to a compound having the followingstructure (IIi):

wherein:

M¹ is a moiety comprising three or more aryl or heteroaryl rings, orcombinations thereof;

R¹ is H, C₁-C₆ alkyl or alkoxy;

R² is cyanoalkyl;

R⁶ is C₁-C₆ alkyl; and

L¹ and L⁴ are each independently optional alkylene or heteroalkylenelinkers.

In some embodiments of (Ii), each R⁶ is isopropyl. In some embodiments,R² is 2-cyanoethyl. In other embodiments, R² is 2-cyanoethyl. In evenother embodiments, R¹ is H. In yet more embodiments, L¹ and L⁴ are eachindependently alkylene linkers, such as methylene linkers.

M¹ is generally a visually detectable moiety or substance. For example,M¹ may be visually detectable in the UV, visible or IR spectrum. In someof any of the foregoing, M¹ is, at each occurrence, independentlyfluorescent or colored. For example, in some embodiments M¹ isfluorescent.

In certain embodiments, M¹ is not a purine or pyrimidine base, such as,but not limited to guanine, cytosine, thymidine and adenine. In otherembodiments, M¹ is not a porphyrin. In other embodiments, M¹ is not oneof the following:

In still other embodiments of any of the foregoing, M¹ comprises threeor more aryl or heteroaryl rings, or combinations thereof, for examplefour or more aryl or heteroaryl rings, or combinations thereof, or evenfive or more aryl or heteroaryl rings, or combinations thereof. In someembodiments, M¹ comprises six aryl or heteroaryl rings, or combinationsthereof. In further embodiments, the rings are fused. For example insome embodiments, M¹ comprises three or more fused rings, four or morefused rings, five or more fused rings, or even six or more fused rings.

In some embodiments, M¹ is cyclic. For example, in some embodiments M¹is carbocyclic. In other embodiment, M¹ is heterocyclic. In still otherembodiments of the foregoing, M¹, at each occurrence, independentlycomprises an aryl moiety. In some of these embodiments, the aryl moietyis multicyclic. In other more specific example, the aryl moiety is afused-multicyclic aryl moiety, for example which may comprise at least3, at least 4, or even more than 4 aryl rings.

In other embodiments of any of the foregoing compounds of structure (I),M¹, at each occurrence, independently comprises at least one heteroatom.For example, in some embodiments, the heteroatom is nitrogen, oxygen orsulfur.

In still more embodiments of any of the foregoing, M¹, at eachoccurrence, independently comprises at least one substituent. Forexample, in some embodiments the substituent is a fluoro, chloro, bromo,iodo, amino, alkylamino, arylamino, hydroxy, sulfhydryl, alkoxy,aryloxy, phenyl, aryl, methyl, ethyl, propyl, butyl, isopropyl, t-butyl,carboxy, sulfonate, amide, or formyl group.

In some even more specific embodiments of the foregoing, M¹, at eachoccurrence, independently is a dimethylaminostilbene, quinacridone,fluorophenyl-dimethyl-BODIPY, his-fluorophenyl-BODIPY, acridine,terrylene, sexiphenyl, porphyrin, benzopyrene,(fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl,(bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl,bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthracyl,squaraine, squarylium, 9,10-ethynylanthracene or ter-naphthyl moiety. Inother embodiments, M¹ is, at each occurrence, independently p-terphenyl,perylene, azobenzene, phenazine, phenanthroline, acridine,thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, orperylene amide or a derivative thereof. In still more embodiments, M¹is, at each occurrence, independently a coumarin dye, resorufin dye,dipyrrometheneboron difluoride dye, ruthenium bipyridyl dye, energytransfer dye, thiazole orange dye, polymethine orN-aryl-1,8-naphthalimide dye.

In still more embodiments of any of the foregoing, M¹ at each occurrenceis the same. In other embodiments, each M¹ is different. In still moreembodiments, one or more M¹ is the same and one or more M¹ is different.

In some embodiments, M¹ is pyrene, perylene, perylene monoimide or 6-FAMor derivative thereof. In some other embodiments, M¹ has one of thefollowing structures:

In some embodiments, M² is selected from any one of the above describedM¹ moieties. In some embodiments, M¹ and M² are the same. In otherembodiments, M¹ and M² are different.

In other embodiments, at least one occurrence of M² is a base pairingmoiety. For example, in some embodiments each occurrence of M² is a basepairing moiety. In some of these embodiments, the base pairing moiety isa purine, a pyrimidine, a dihydropyrimidine or a derivative thereof. Infurther embodiments, the base pairing moiety has one of the followingstructures:

In some of the foregoing embodiments, R² is a linker comprising acovalent bond to a biomolecule or microparticle, and R³ is H, OH,phosphate, thiophosphate, phosphoalkyl, phosphoalkylether,thiophosphoalkyl or thiophosphoalkylether. For example, in someembodiments R² is a linker comprising a covalent linkage to abiomolecule. For example, a nucleic acid, amino acid or a polymerthereof (e.g., polynucleotide or polypeptide). In still moreembodiments, the biomolecule is an enzyme, receptor, receptor ligand,antibody, glycoprotein, aptamer or prion.

In yet other embodiments, R² is a linker comprising a covalent linkageto a microparticle. For example, in some embodiments the microparticleis a polymeric bead or nonpolymeric bead.

In some more embodiments of some of the foregoing embodiment, R⁷, R⁸,R⁹, R¹⁰, R¹¹ and R¹², when present, are each H.

In some other embodiments, x, y and z are each 1.

In some of any of the foregoing embodiments, R³ is —OH, phosphate orthiophosphate. In other embodiments, R³ is —OH, phosphate,thiophosphate, phosphoalkyl or thiophosphoalkyl. In some moreembodiments, R³ is phosphoalkyl or thiophosphoalkyl.

The present invention includes monomeric compounds (e.g., n=1) as wellas oligomeric compound (e.g., n is 2-20 or 2-10). In other of theforegoing embodiments, n is an integer from 1 to 5. For example, in someembodiments, n is an integer from 2 to 10, or 2 to 5, such as 3. Inother embodiments n is 1. In more embodiments, n is 2. In otherembodiments n is 3. In more embodiments, n is 4. In other embodiments nis 5. In more embodiments, n is 6. In other embodiments n is 7. In moreembodiments, n is 8. In other embodiments n is 9. In more embodiments, nis 10.

Any number of methylene spacer units (i.e., x, y and z) can be included.In some embodiments x is 0. In other embodiments x is 1. In moreembodiments, x is 2. In some embodiments x is 3. In other embodiments xis 4. In more embodiments, x is 5.

In some embodiments y is 0. In other embodiments y is 1. In moreembodiments, y is 2. In some embodiments y is 3. In other embodiments yis 4. In more embodiments, y is 5.

In some embodiments z is 1-5. In some embodiments z is 0. In otherembodiments z is 1. In more embodiments, z is 2. In some embodiments zis 3. In other embodiments z is 4. In more embodiments, z is 5.

In other embodiments, x is 1, y is 0 and z is 1. In other embodiments, xis 0, y is 1 and z is 1.

In some specific embodiments, a compound having one of the followingstructures is provided:

For ease of illustration, various compounds comprising phosphorousmoieties (e.g., phosphate and the like) are depicted in the anionicstate (e.g., —OPO₃ ²⁻). One of skill in the art will readily understandthat the charge is dependent on pH and the uncharged (e.g., protontatedor salt, such as sodium or other cation) forms are also included in thescope of the invention.

Compositions comprising any of the foregoing compounds and one or morebiomolecules are provided in various other embodiments. In someembodiments, use of such compositions in analytical methods fordetection of the one or more biomolecules are also provided.

In still other embodiments, the compounds are useful in variousanalytical methods. For example, in certain embodiments the disclosureprovides a method of staining a sample, the method comprising adding tosaid sample a compound of structure (I), wherein R² is a linkercomprising a covalent bond to a biomolecule or microparticle, and R³ isH, OH, phosphate, thiophosphate, phosphoalkyl, phosphoalkylether,thiophosphoalkyl or thiophosphoalkylether in an amount sufficient toproduce an optical response when said sample is illuminated at anappropriate wavelength.

In some embodiments of the foregoing methods, R² is a linker comprisinga covalent linkage to a biomolecule. For example, a nucleic acid, aminoacid or a polymer thereof (e.g., polynucleotide or polypeptide). Instill more embodiments, the biomolecule is an enzyme, receptor, receptorligand, antibody, glycoprotein, aptamer or prion.

In yet other embodiments of the foregoing method, R² is a linkercomprising a covalent linkage to a microparticle. For example, in someembodiments the microparticle is a polymeric bead or nonpolymeric bead.

In even more embodiments, said optical response is a fluorescentresponse.

In other embodiments, said sample comprises cells, and some embodimentsfurther comprise observing said cells by flow cytometry.

In still more embodiments, the method further comprises distinguishingthe fluorescence response from that of a second fluorophore havingdetectably different optical properties.

In other embodiments, the disclosure provides a method for visuallydetecting a biomolecule, comprising:

-   -   (a) providing a compound of structure (I), wherein R² is a        linker comprising a covalent bond to the biomolecule, and R³ is        H, OH, phosphate, thiophosphate, phosphoalkyl,        phosphoalkylether, thiophosphoalkyl or thiophosphoalkylether;        and    -   (b) detecting the compound by its visible properties.

For example, a nucleic acid, amino acid or a polymer thereof (e.g.,polynucleotide or polypeptide). In still more embodiments, thebiomolecule is an enzyme, receptor, receptor ligand, antibody,glycoprotein, aptamer or prion.

In other embodiments, a method for visually detecting a biomolecule isprovided, the method comprising:

-   -   (a) admixing any of the foregoing compounds with one or more        biomolecules; and    -   (b) detecting the compound by its visible properties.

It is understood that any embodiment of the compounds of structure (I),as set forth above, and any specific choice set forth herein for a R¹,R², R³, L¹, L², L³, L⁴, L⁵, L⁶, M¹, M², A, q, w or n variable in thecompounds of structure (I), as set forth above, may be independentlycombined with other embodiments and/or variables of the compounds ofstructure (I) to form embodiments of the inventions not specifically setforth above. In addition, in the event that a list of choices is listedfor any particular R or M group in a particular embodiment and/or claim,it is understood that each individual choice may be deleted from theparticular embodiment and/or claim and that the remaining list ofchoices will be considered to be within the scope of the invention.

It is understood that in the present description, combinations ofsubstituents and/or variables of the depicted formulae are permissibleonly if such contributions result in stable compounds.

It will also be appreciated by those skilled in the art that in theprocess described herein the functional groups of intermediate compoundsmay need to be protected by suitable protecting groups. Such functionalgroups include hydroxy, amino, mercapto and carboxylic acid. Suitableprotecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl(for example, t-butyldimethylsilyl, t-butyldiphenylsilyl ortrimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitableprotecting groups for amino, amidino and guanidino includet-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protectinggroups for mercapto include —C(O)—R″ (where R″ is alkyl, aryl orarylalkyl), p-methoxybenzyl, trityl and the like. Suitable protectinggroups for carboxylic acid include alkyl, aryl or arylalkyl esters.Protecting groups may be added or removed in accordance with standardtechniques, which are known to one skilled in the art and as describedherein. The use of protecting groups is described in detail in Green, T.W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rdEd., Wiley. As one of skill in the art would appreciate, the protectinggroup may also be a polymer resin such as a Wang resin, Rink resin or a2-chlorotrityl-chloride resin.

Furthermore, all compounds of the invention which exist in free base oracid form can be converted to their salts by treatment with theappropriate inorganic or organic base or acid by methods known to oneskilled in the art. Salts of the compounds of the invention can beconverted to their free base or acid form by standard techniques.

The following Reaction Schemes illustrate exemplary methods of makingcompounds of this invention. It is understood that one skilled in theart may be able to make these compounds by similar methods or bycombining other methods known to one skilled in the art. It is alsounderstood that one skilled in the art would be able to make, in asimilar manner as described below, other compounds of structure (I) notspecifically illustrated below by using the appropriate startingcomponents and modifying the parameters of the synthesis as needed. Ingeneral, starting components may be obtained from sources such as SigmaAldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI,and Fluorochem USA, etc. or synthesized according to sources known tothose skilled in the art (see, for example, Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, 5th edition (Wiley, December2000)) or prepared as described in this invention.

Reaction Scheme I illustrates and exemplary method for preparingcompounds of structure I. Referring to Reaction Scheme 1, compounds ofstructure a can be purchased or prepared by methods well-known to thoseof ordinary skill in the art. Reaction of a with M¹-X, where x is ahalogen such as bromo, under Suzuki coupling conditions known in the artresults in compounds of structure b. Compounds of structure b can bemodified to obtain number of other compounds of structure I. Forexample, compounds of structure b can be oligomerized to obtain othercompounds of structure I (i.e., where n is greater than 1, such as2-10). Exemplary methods for oligomerization include methods analogousto phosphoramadite-based solid-phase oligonucleotide synthesis, which iswell known in the art.

Reaction Scheme II illustrates an alternative method for preparation ofcompounds of structure I. In this approach, a compound of structure c,which can be purchased or prepared by well-known techniques, is reactedwith M¹-Z to yield compounds of structure d. Here, Y and Z representfunction groups having complementary reactivity (i.e., functional groupswhich react to form a covalent bond). Z may be pendant to M¹ or a partof the structural backbone of M, for example a cyclic anhydride. Y maybe any number of functional groups, such as amino.

Compounds of structure (I) comprising ribose moieties are preparedaccording to analogous procedures or purchased from commercial sources(e.g., as phosphoramadites).

In certain embodiments, the compounds of structure I are oligomerscomprising from 2-10 repeating units. Such oligomers can be preparedusing methods analogous to well-known automated DNA synthesis methods.DNA synthesis methods are well-known in the art. Briefly, two alcoholgroups are functionalized with a dimethoxytrityl (DMT) group and a2-cyanoethyl-N,N-diisopropylamino phosphoramidite group, respectively.The phosphoramidite group is coupled to an alcohol group, typically inthe presence of an activator such as tetrazole, followed by oxidation ofthe phosphorous atom with iodine. The dimethoxytrityl group can beremoved with acid (e.g., chloroacetic acid) to expose the free alcohol,which can be reacted with a phosphoramidite group. The 2-cyanoethylgroup can be removed after oligomerization by treatment with aqueousammonia.

Preparation of the phosphoramidites used in the oligomerization methodsis also well-known in the art. For example, a primary alcohol (e.g., R³)can be protected as a DMT group by reaction with DMT-Cl. A secondaryalcohol (e.g., R²) is then functionalized as a phosphoramidite byreaction with an appropriate reagent such as 2-cyanoethylN,N-dissopropylchlorophosphoramidite. Methods for preparation ofphosphoramidites and their oligomerization are well-known in the art anddescribed in more detail in the examples.

The following examples are provided for purposes of illustration, notlimitation.

EXAMPLES General Methods

¹H and ³¹P NMR spectra were obtained on a JEOL 400 MHz spectrometer. ³¹PNMR spectra were referenced against 85% aqueous phosphoric acid and ¹Hspectra were referenced against TMS. Reverse phase HPLC dye analysis wasperformed using a Waters Acquity UHPLC system with a 2.1 mm×50 mmAcquity BEH-C18 column held at 45° C. Mass spectral analysis wasperformed on a Waters/Micromass Quattro micro MS/MS system (in MS onlymode) using MassLynx 4.1 acquisition software. Mobile phase used forLC/MS on dyes was 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), 8.6mM triethylamine (TEA), pH 8. Phosphoramidites and precursor moleculeswere analyzed using an Agilent Infinity 1260 UHPLC system with a diodearray detector and High Performance Autosampler using an Aapptec©Spirit™ Peptide C18 column (4.6 mm×100 mm, 5 μm particle size).Excitation and emission profiles experiments were recorded on a CaryEclipse spectra photometer.

All reactions were carried out in oven dried glassware under a nitrogenatmosphere unless otherwise stated. Commercially available DNA synthesisreagents were purchased from Glen Research (Sterling, Va.) Anhydrouspyridine, toluene, dichloromethane, diisopropylethyl amine,triethylamine, acetic acid, pyridine, and THF were purchased fromAldrich. All other chemicals were purchased from Aldrich or TCI and wereused as is with no additional purification.

All oligomer dyes were synthesized on an ABI 394 DNA synthesizer usingstandard protocols for the phosphoramidite-based coupling approach. Thechain assembly cycle for the synthesis of oligomers was the following:(i) detritylation, 3% trichloroaceticacid in dichloromethane, 1 min;(ii) coupling, 0.1 M phosphoramidite and 0.45 M tetrazole inacetonitrile, 10 min; (iii) capping, 0.5 M acetic anhydride inTHF/lutidine, 1/1, v/v 15 s; (iv) oxidation, 0.1 M iodine inTHF/pyridine/water, 10/10/1, v/v/v, 30 s.

Chemical steps within the cycle were followed by acetonitrile washingand flushing with dry argon for 0.2-0.4 min. Cleavage from the supportand removal of base and phosphoramidate protecting groups was achievedby treatment with ammonia for 1 hour at room temperature. Oligomer dyeswere then analyzed by reverse phase HPLC as described above.

Example 1 Synthesis of Phosphoramidite Dye Monomers

1-O-(4,4′-dimethoxytrityl-2-methylene-1,3-propanediol(1). Into a dry 500mL round bottom flask was put a stir bar. After flushing with nitrogen,dry pyridine (240 mL) was added, and the flask was cooled in an ice bathfor 15 minutes. Upon cooling DMTrCl (7.65 g, 22.5 mmol) was added afterwhich the flask was stirred overnight in a refrigerator at 4° C. under anitrogen atmosphere. Several drops of methanol were then added and thereaction was concentrated in vacuo to a viscous gum. The resulting gumwas dissolved in EtOAc (200 mL) and washed with NaHCO₃ (250 mL) and sat.NaCl (250 mL). The organic layer was dried over Na₂SO₄ and concentratedin vacuo to a viscous gum. The isolated crude product wash then purifiedby silica gel column chromatography eluting with a gradient ofEtOAc:hexanes (25:75 v/v)-(1:1 v/v) to give 1 as a clear gum (5.21g,60%). ¹H NMR was recorded and found to be consistent with the structureof compound 1.

1-O-(4,4′-dimethoxytrityl)-2-hydroxymethyl-3-pyrenylpropanol(2). Into adry 250 mL round bottom flask fitted with a condenser was put a stirbar. The flask was purged with nitrogen, and dry THF (40 mL) andcompound 1 (5.0 g, 12.8 mmol) were added. 0.5 M 9-BBN in THF (65 mL, 32mmol) was added via syringe and the reaction was heated to reflux for 12hrs. After allowing the reaction to cool to room temperature, 3M K₂CO₃(11 ml) and dry DMF (100 mL) were added. 1-Bromopyrene (2.0 g, 6.5 mmol)and PdCl₂(dppf) (0.65 g, 0.8 mmol) were added, and the solution wasallowed to stir for 15 hrs at room temperature. The reaction mixture waspoured into CH₂Cl₂ (300 mL) and washed with H₂O (500 mL). The aqueouslayer was then back extracted with additional CH₂Cl₂ (200 mL). Thecombined organic layers were washed with sat. NaCl (300 mL), dried overNa₂SO₄, and concentrated in vacuo to a viscous gum. The isolated crudeproduct wash then purified by silica gel column chromatography elutingwith a gradient of EtOAc:hexanes (25:75 v/v)-(1:1 v/v) to give 2 as aclear gum (3.0 g, 79%). The ¹H NMR spectrum was recorded and found to beconsistent with the structure of compound 2.

1-O-(4,4′-dimethoxytrityl)-2-methylpyrene-3-O-(2-cyanoethyl-N,N-diisopropyl)propanephosphoramidite (3). Into a dry 100 mL round bottom flask was put a stirbar. After purging the flask with nitrogen, CH₂Cl₂ (20 mL) and compound2 (0.30 g, 0.50 mmol) were added. N,N-Diisopropylethylamine (0.88 mL,5.0 mmol) and 2-cyanoethyl diisopropychlorophosphoramidite (0.45 mL, 2.0mmol) were added via syringe. After 1 hour of stirring at roomtemperature, the reaction was determined to be complete by TLC analysis.The crude reaction mixture was then purified directly by silica gelcolumn chromatography eluting with a gradient of EtOAc:hexanes:TEA(22.5:72.5:5 v/v/v) to give 3 as a white foam (0.28 g, 70%). The ³¹P NMRspectrum was recorded and found to be consisted with the structure ofcompound 3: Purity was determined by HPLC analysis with detection at 254and 340 nm.

Other compounds with different Ar groups (e.g., perylene) were preparedin an analogous manner.

Example 2 Synthesis of Perylene Carbodiimide Dye Monomer

N-(2,3-propanediol) perylenemonoimide(4). Into a dry 200 mL round bottomflask fitted with a condenser was put a stir bar and perylenemonoanhydride^(l) (1.83 g, 5.67 mmol). After adding3-amino-1,2-propanediol (1.1 g, 2.1 mmol) and imidazole (14.3 g, 0.21mol), the vessel was heated to 140° C. in an oil bath for 15 hours. Thereaction was allowed to cool to room temperature and then 10% HCl wasadded (500 mL). The resulting deep red precipitate was collected byfiltration, washed well with water and dried at 180° C. for severalhours to yield 4 as a deep red solid (1.95 g, 86%).

N-(3-O-(4,4′-dimethoxytrityl-2-hydroxypropane) perylenemonoimide(5).Into a dry 200 mL round bottom flask was put a stir bar. After purgingthe flask with nitrogen, dry pyridine (120 mL), compound 4 (0.44 g, 1.1mmol), and dimethoxytritylchloride (0.45 g, 1.3 mmol) were all added,and the reaction was allowed to stir at room temperature for 48 hours.Several drops of methanol were then added, and the reaction wasconcentrated in vacuo to a viscous gum. The resulting gum was dissolvedin CH₂Cl₂ (200 mL) and washed with sat. NaCl (200 mL). The aqueous layerwas washed with in CH₂Cl₂ (3×100 mL). The combined organic layers weredried over Na₂SO₄ and concentrated in vacuo to a viscous gum. Theisolated crude product wash then purified by silica gel columnchromatography eluting with a gradient of EtOAc:CH₂Cl₂ (0:100 v/v)-(2:3v/v) to give 5 as a red foam (0.25 g, 50%).

N-(3-O-(4,4′-dimethoxytrityl-2-O-(2-cyanoethyl-N,N-diisopropylaminophosphoramidite)perylene-monoimide (6). Into a dry 50 mL round bottomflask was put a stir bar. After purging the flask with nitrogen, CH₂Cl₂(5 mL) and compound 5 (0.25 g, 0.36 mmol) were added.N,N-diisopropylethylamine (0.24 mL, 1.79 mmol) and 2-cyanoethylN,N-diisopropychlorophosphoramidite (0.16 mL, 0.72 mmol) were added viasyringe. After 1 hour of stirring at room temperature, the reaction wasdetermined to be complete by TLC analysis. The crude reaction mixturewas then purified directly by silica gel column chromatography elutingwith CH₂Cl₂:TEA (95:5 v/v) to give 6 as a dark red foam (0.26 g, 80%).The purified compound was analyzed by RP-HPLC with observation at 254and 500 nm. Two diastereomers were found to be present.

Example 3 Synthesis of Oligomer Dyes

Oligomer dyes were synthesized on an Applied Biosystems 394 DNA/RNAsynthesizer or on GE AKTÄ 10 OligoPilot on either 1 μmol or 10 μmolscales and possessed a 3′-phosphate group. Dyes were synthesizeddirectly on CPG beads or on polystyrene solid support. The dyes weresynthesized in the 3′ to 5′ direction by standard solid phase DNAmethods. Coupling methods employed standard β-cyanoethyl phosphoramiditechemistry conditions. All phosphoramidite monomers were dissolved inacetonitrile/dichloromethane (0.1 M solutions), and were added insuccessive order using the following synthesis cycles: 1) removal of the5′-dimethoxytrityl protecting group with dichloroacetic acid in toluene,2) coupling of the next phosphoramidite with activator reagent inacetonitrile, 3) oxidation with iodine/pyridine/water, and 4) cappingwith acetic anhydride/1-methylimidizole/acetonitrile. The synthesiscycle was repeated until the 5′ Oligofloroside was assembled. At the endof the chain assembly, the monomethoxytrityl (MMT) group ordimthoxytrityl (DMT) group was removed with dichloroacetic acid indichloromethane or dichloroacetic acid in toluene. The dyes were cleavedfrom the solid support using concentrated aqueous ammonium hydroxide atroom temperature for 2-4 hours. The product was concentrated in vacuoand Sephadex G-25 columns were used to isolate the main product whichwas analyzed by RP-HPLC. Sequences, of representative oligomers preparedaccording to the general method, as well as spectral properties andmolecular weights (MW) determined by electrospray mass spectrometry arepresented in Table 1.

TABLE 1 Representative Oligomer Dyes and TheirObserved Masses and Optical Properties Calcu- λ_(max) λ_(max) latedObserved (Exc.) (Em.) Sequence Mass Mass nm nm 5′-FC_(S)F-3′ 1277.21276.8 492 519 5′-C_(S)C_(S)C_(S)YYY- 1447.2 1447.5 330, 376, 3′ 344395, 481 5′-IC_(S)C_(S)-3′ 777.2 777.6 263, 581 4945′-C_(S)C_(S)IC_(S)C_(S)- 1024.4 1024.4 263, 581 3′ 490 5′-amino-C_(S)1191.5 1192.9 263, 581 C_(S)IC_(S)C_(S)-3′ 494 5′-IC_(S)C_(S)C_(S)1271.6 1272.9 263, 581 C_(S)C_(S)C_(S)-3′ 494 5′-EC_(S)C_(S)[SS] 1648.81648.8 418, 453, C_(S)C_(S)E-3′ 446 482 5′-Y-3′ 370.8 371.2 326, 377,342 397 5′-YY-3′ 723.6 723.5 327, 376, 343 396, 484 5′-YYYY- 1429.21429.2 328, 376, 3′ 343 397, 478 5′-YYYYY- 1782.0 1783.9 328, 376, 3′344 397, 478 5′-YYYYYY- 2134.8 2132.0 328, 376, 3′ 344 397, 4785′-YT_(h)YT_(h)Y- 1688.8 1688.9 328, 376, 3′ 344 397, 4845′-YT_(h)T_(h)YT_(h) 2301.2 2303.6 328, 376, T_(h)Y-3′ 344 397, 4865′-YT_(h)T_(h)T_(h) 2913.6 2917.1 328, 377, YT_(h)T_(h)T_(h)Y-3′ 344396, 485 5′-YAYAY-3′ 1702.8 1703.1 328, 377, 344 396, 485 5′-YAAA-3′1310.4 1311.0 327, 377, 343 397 5′-YAAAYAA 2602.8 2604.8 329, 377, A-3′346 397 5′-YAAAAYA 3582.0 3581.0 329, 378, AAAY-3′ 345 399 5′-YAAAAAY4208.4 4208.0 329, 378, AAAAAY-3′ 345 398 5′-YAAAYAA 4248.0 4247.0 329,378, AYAAAY-3′ 345 398

Structures for the above sequences are illustrated herein above.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification, including butlimited to U.S. Patent Application No. 61/868,973, filed Aug. 22, 2013,are incorporated herein by reference, in their entirety to the extentnot inconsistent with the present description.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A compound having the following structure (III):

wherein: M¹ and M² are, at each occurrence, independently a moietycomprising two or more double bonds and at least one degree ofconjugation, and at least one occurrence of M¹ is a moiety comprisingthree or more aryl or heteroaryl rings, or combinations thereof; L¹, L³,L⁴ L⁶, L⁷ and L⁸ are, at each occurrence, independently optionalalkylene or heteroalkylene linkers; R¹ is, at each occurrence,independently H, alkyl or alkoxy; R² is an electron pair, H, alkyl,alkylether, hydroxylalkyl, aminoalkyl, hydroxylalkylether,sulfhydrylalkyl, sulfyhdrylalkylether, cyanoalkyl, phospho, thiophospho,alkylphospho, alkylthiophospho, alkyletherphospho,alkyletherthiophospho, phosphoalkyl, phosphoalkylether, thiophosphoalkylor thiophosphoalkylether, or R² is a linker comprising a covalent bondto a biomolecule or microparticle; R³ is H, OH, SH, —NH₂, alkyl,alkylether, hydroxylalkyl, aminoalkyl, hydroxylalkylether,sulfhydrylalkyl, sulfyhdrylalkylether, cyanoalkyl, —Oaralkyl, phosphate,thiophosphate, alkylphospho, alkylthiophospho, —Oalkylphospho,—Oalkylthiophospho, alkyletherphospho, alkyletherthiophospho,—Oalkyletherphospho, —Oalkyletherthiophospho phosphoalkyl,phosphoalkylether, thiophosphoalkyl, thiophosphoalkylether,—Ophosphoalkyl, O-phosphoalkylether, —Othiophosphoalkyl or—Othiophosphoalkylether; R⁴ is, at each occurrence, independently O⁻,S⁻, OZ, SZ or N(R⁶)₂, where Z is a cation and each R⁶ is independently Hor alkyl; R⁵ is, at each occurrence, independently oxo, thioxo orabsent; n is an integer from 2 to 20; and q and w are each independently0 or 1 for each integral value of n, wherein q is 1 for at least twointegral values of n, or wherein q and w are each independently one forat least one integral value of n.
 2. (canceled)
 3. The compound of claim1, wherein the compound has the following structure (IIa):

wherein: M¹ is, at each occurrence, independently a moiety comprisingtwo or more double bonds and at least one degree of conjugation, and atleast one occurrence of M¹ is a moiety comprising four or more aryl orheteroaryl rings, or combinations thereof; L¹, L⁷ and L³ are, at eachoccurrence, independently optional alkylene or heteroalkylene linkers;R¹ is, at each occurrence, independently H, alkyl or alkoxy; R² is H,alkyl, alkylether, hydroxylalkyl, aminoalkyl, hydroxylalkylether,sulfhydrylalkyl, sulfyhdrylalkylether, cyanoalkyl, phospho, thiophospho,alkylphospho, alkylthiophospho, alkyletherphospho,alkyletherthiophospho, phosphoalkyl, phosphoalkylether, thiophosphoalkylor thiophosphoalkylether, or R² is a linker comprising a covalent bondto a biomolecule or microparticle; R³ is H, OH, SH, —NH₂, alkyl,alkylether, hydroxylalkyl, aminoalkyl, hydroxylalkylether,sulfhydrylalkyl, sulfyhdrylalkylether, cyanoalkyl, —Oaralkyl, phosphate,thiophosphate, alkylphospho, alkylthiophospho, —Oalkylphospho,—Oalkylthiophospho, alkyletherphospho, alkyletherthiophospho,—Oalkyletherphospho, —Oalkyletherthiophospho phosphoalkyl,phosphoalkylether, thiophosphoalkyl, thiophosphoalkylether,—Ophosphoalkyl, O-phosphoalkylether, —Othiophosphoalkyl or—Othiophosphoalkylether; R⁴ is, at each occurrence, independently O⁻,S⁻, OZ, SZ or N(R⁶)₂, where Z is a cation and each R⁶ is independently Hor alkyl; R⁵ is, at each occurrence, independently oxo, thioxo orabsent; and n is an integer from 2 to
 10. 4. (canceled)
 5. The compoundof claim 4, wherein the compound has one of the following structures(IIc), (IId), (IIe) or (IIf):


6. (canceled)
 7. The compound of claim 1, wherein the compound has thefollowing structure (IIIb):

wherein: R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are, at each occurrence,independently H or alkyl; and x¹, x², y¹, y², z¹ and z² are, at eachoccurrence, independently an integer from 0 to
 5. 8-10. (canceled) 11.The compound of claim 14, wherein R² has one of the followingstructures:

wherein: R^(2a) is —OH, —NH₂, or —SH; and a is an integer from 1 to 10.12. (canceled)
 13. The compound of claim 1, wherein R² has one of thefollowing structures:

wherein: R^(2a) is —OH, —SH, —NH₂, phosphate or thiophosphate; R^(4a)and R^(4b) are independently O⁻, S⁻, OZ or SZ, where Z is a cation;R^(5a) and R^(5b) are independently oxo, or thioxo; and a, b and c areeach independently integers from 1 to
 10. 14. The compound of claim 1,wherein R³ is OH.
 15. (canceled)
 16. The compound of claim 1, wherein R³has one of the following structures:

wherein: R^(3a) is —OH, —SH, —NH₂, phosphate or thiophosphate; R^(4a)and R^(4b) are independently O⁻, S⁻, OZ or SZ, where Z is a cation;R^(5a) and R^(5b) are independently oxo, or thioxo; and b and c are eachindependently integers from 1 to
 10. 17-28. (canceled)
 29. A compoundhaving the following structure (IV):

wherein: M¹ is, at each occurrence, independently a moiety comprisingtwo or more double bonds and at least one degree of conjugation, and atleast one occurrence of M¹ is a moiety comprising three or more aryl orheteroaryl rings, or combinations thereof; L¹, L³, and L⁷ are, at eachoccurrence, independently optional alkylene or heteroalkylene linkers;R¹ is, at each occurrence, independently H, alkyl or alkoxy; R² isphospho, thiophospho, alkylphospho, alkylthiophospho, alkyletherphospho,alkyletherthiophospho, phosphoalkyl, phosphoalkylether, thiophosphoalkylor thiophosphoalkylether, or R² is a linker comprising a covalent bondto a biomolecule or microparticle; R³ is H, OH, SH, —NH₂, alkyl,alkylether, hydroxylalkyl, aminoalkyl, hydroxylalkylether,sulfhydrylalkyl, sulfyhdrylalkylether, phosphate, thiophosphate,alkylphospho, alkylthiophospho, —Oalkylphospho, —Oalkylthiophospho,alkyletherphospho, alkyletherthiophospho, —Oalkyletherphospho,—Oalkyletherthiophospho phosphoalkyl, phosphoalkylether,thiophosphoalkyl, thiophosphoalkylether, —Ophosphoalkyl,O-phosphoalkylether, —Othiophosphoalkyl or —Othiophosphoalkylether; R⁴is, at each occurrence, independently O⁻, S⁻, OZ, SZ or N(R⁶)₂, where Zis a cation and each R⁶ is independently H or alkyl; R⁵ is, at eachoccurrence, independently oxo, thioxo or absent; n is an integer from 1to
 20. 30-44. (canceled)
 45. A compound having the following structure(Ih):

wherein: M¹ is a moiety comprising three or more aryl or heteroarylrings, or combinations thereof; R¹ is H, alkyl or alkoxy: R² is H, anelectron pair or a cation; R³ is OH; R⁴ is O⁻, S⁻, OZ, SZ where Z is acation; R⁵ is oxo or thioxo; R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are, at eachoccurrence, independently H or alkyl; and x, y and z are, at eachoccurrence, independently an integer from 0 to
 5. 46-47. (canceled) 48.The compound of claim 1, wherein at least one M¹ is a moiety comprisingfour or more aryl or heteroaryl rings, or combinations thereof.
 49. Thecompound of claim 1, wherein M¹ is, at each occurrence, independentlyfluorescent or colored. 50-60. (canceled)
 61. The compound of claim 1,wherein M¹ has one of the following structures:

62-65. (canceled)
 66. The compound of claim 1, wherein R² is a linkercomprising a covalent bond to a biomolecule or microparticle, and R³ isH, OH, phosphate, thiophosphate, phosphoalkyl, phosphoalkylether,thiophosphoalkyl or thiophosphoalkylether. 67-81. (canceled)
 82. Acompound having one of the following structures:


83. A method of staining a sample, comprising adding to said sample thecompound of claim 661 in an amount sufficient to produce an opticalresponse when said sample is illuminated at an appropriate wavelength.84-87. (canceled)
 88. A method for visually detecting a biomolecule, themethod comprising: (a) providing the compound of claim 66; and (b)detecting the compound by its visible properties.
 89. A method forvisually detecting a biomolecule, the method comprising: (a) admixingthe compound of claim 1 with one or more biomolecules; and (b) detectingthe compound by its visible properties.
 90. A composition comprising thecompound of claim 1 and one or more biomolecules.
 91. (canceled)